Gliederung

Objective: Clinical transplantation of neuronal precursors has been shown to alter the progression of Huntington's disease (HD). Due to the limited availability of transplantable tissue, strategies to enhance the striatal population of fetal precursor cells are needed. In this study, the impact of transient transfection of two proneuronal transcription factors, Mash1 and Helt, on differentiation and maturation of ganglionic eminence-derived precursors cells is investigated.

Methods: Fetal ganglionic eminences (lateral and medial) were dissected from rat embryos on E13.5, E14.5 and E15.5. Coding regions of Mash1 and rat Helt were cloned into an expression vector under transcriptional control of elongation factor 1 alpha. Cells were grown adherent and chemically transfected for 24 hrs. Transfected cells were selected, and cultured for 7 days, and afterwards processed for immunocytochemistry.

Results: Data indicate that the expression of the transcription factors lasts about 3 to 4 days. Double transfection with Helt and Mash1 significantly increases the number of neuronal precursors (Nestin), as well as pre-mature and mature neurons (Tuj1 and Map2a/b). Furthermore, a drastic increase of DARPP-32 positive cells is observed after double transfection compared to single transfection. In addition, double transfection slightly increases the differentiation of astrocytes, but inhibits generation of oligodentrocytes. Semi-qRT-PCR reveals an increase in striatal markers on a transcriptional level 3 days after transfection already.

Conclusions: We conclude that transient transfection provides a non-hazard method for genetic manipulation of neural precursors. Transient expression of Mash1 and Helt is sufficient to increase neurogenesis and astrogenesis, and further to induce a striatal-like phenotype in vitro.